The focus of this interdisciplinary research-training proposal is the analysis of HIV envelope glycoprotein (gp120) glycosylation. This protein is highly glycosylated (22-25 putative glycosylation sites depending on strain) and binds to CD4 on host cells to initiate viral entry. As with many glycosylated proteins, a variety of different polysaccharides are attached to the protein's Asn residues. Although this process is enzymatic in nature, proteins are not necessarily homogenously glycosylated such that individual sites may contain a wide array of glycan moieties. By studying the similarities and differences between gp120 from live virus cultures derived from infective molecular clones of transmitted HIV, we aim to tease apart the relative importance of the glycosylation pattern of gp120 and its relationship with immunological avoidance, structural folding and infectivity. Although much is known regarding the importance of individual glycosylation sites through previous selective mutation studies in regard to immune avoidance and infectivty, the analysis of specific glycosylation patterns (identifying each glycan and its location on the protein) has been difficult to study. However, recent advances in mass spectrometric methods to study glycans provide a means to study the chemical nature of the specific glycans bound to specific amino acids in the proteins. The proposed research will adapt these glycomic and glycoproteomic methods to the study of the highly glycosylated gp120 protein from HIV. Specifically, we will study the glycosylation patterns of HIV gp120 with mass spectrometry to identify homogenously and heterogeneously glycosylated sites and the complement of glycan structures present at each individual glycosylation site. In addition, we aim to compare glycan incorporation into gp120 from viruses from infectious molecular clones produced from viruses obtained from transmitted viruses as a means to study conserved glycosylation patterns across transmitted HIV strains. An understanding of both the highly variable and highly conserved aspects of glycosylation may provide insight into the role of gp120 glycosylation at specific locations on the protein. While highly heterogeneously glycosylated sites may be associated with immune avoidance, sites that consistently contain the a single or chemical similar glycan moieties may suggest sites for future study as drug or immunological targets.